Gene transfer is the first step of targeted genetic engineering. The commonly used DNA delivery or transformation methods can be categorized into three kinds1. First, certain chemicals such as calcium phosphate and DEAE-dextran are capable of permeabilizing cell membrane and facilitating DNA uptake. Although well established in several model organisms, the chemically facilitated methods suffer from very limited spectrum of applicable species. Second, liposome fusion and viral/phage infection are widely used to infect cells. This type of methods is generally efficient in DNA delivery, but it does not allow control of spatial or temporal specificity of DNA delivery. In addition, viral infection is likely to provoke the host immune response and thus prohibit gene expression and viral re-infection. Third, there are several mechanical methods including microinjection, electroporation, particle bombardment (gene gun) and sonoporation. In general, the mechanical methods are more versatile than the other two kinds, as they are less dependent on cell type. However, microinjection does require certain cell size in order to be performed under microscope; and electroporation, which permeabilizes cell membrane by high-voltage electric fields, is invasive and causes severe damage to cells. Particle bombardment couples a gene to projectiles that penetrate the membrane and hence allows for DNA delivery. However, to date this method is only amenable for surface (e.g. skin) applications. Furthermore, this and all of the aforementioned methods require repeated rounds of washing and other treatments of the cells prior to DNA transformation, making the protocol complex and difficult to be implemented in high-throughput manner.
Sonoporation has also been reported as as a DNA delivery method. Ultrasound is mechanical wave energy at frequencies above 20 kHz, which is inaudible to the human ear. The bioeffects of ultrasound include biomass heating, shear stress and mass transfer, indicating that the sonic energy could be converted into heat or mechanical energy resulting in disruption or relocation of biomass. In addition, ultrasound generates acoustic cavitation in liquid. Ultrasound generates bubbles that grow in the successive acoustic cycles. When the bubbles grow to a critical state, they suddenly collapse and release energy that can damage nearby intact cells or permeabilize cell membrane2. The latter phenomenon, termed reparable sonoporation, has been employed for DNA delivery since it induces temporary pores on the cell membrane for DNA uptake followed by pore resealing and cell survival.
Sonoporation as a DNA delivery method has been employed in animal cells both in vitro and in vivo. For example, 60 seconds of ultrasound exposure efficiently transfect a luciferase reporter plasmid into cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs)3. Furthermore, the short duration of ultrasound exposure caused only mild damage to the cell monolayer and had no impact on the plasmid integrity. More recently, there are mounting studies demonstrating sonoporation as a viable technique to transfect reporter and therapeutic gene constructs into mammalian organs in vivo4. However, there are few studies in non-animal species. Sonoporation is capable of DNA delivery into plant protoplast, suspension cells and intact pieces of plant tissues5. For the budding yeast, one study showed that a low efficiency of 2,000 transformants/microGram DNA was achieved by sonoporation6. For bacteria, there have been three recent studies describing sonoporation protocols for Fusobacterium nucleatum, Escherichia coli, Pseudomonas putida and Pseudomonas fluorescens7-9, all of which are Gram negative bacteria. It remains unclear whether sonoporation is applicable to Gram positive bacteria for gene delivery.